Chemoembolization Composition Comprising Anti-Angiogenic Agents

ABSTRACT

The invention relates to chemoembolization compositions for anti-angiogenic agent delivery. The invention further relates to a method of preparing chemoembolization compositions and to the use of chemoembolization compositions in the method for treating solid tumour cancers.

PRIORITY STATEMENT

This application is a divisonal application of U.S. patent applicationSer. No. 13/989,955 which has a filing date of 7 Aug. 2013, which is anational stage application under 35 U.S.C. §371 of PCT InternationalApplication. No. PCT/IB2011/055360 which has an International filingdate of 29 Nov. 2011, and which claims the benefit under under 35 U.S.C.§119 to Switzerland Application No. 01997/10 filed 29 Nov. 2010. Thecontents of each application recited above are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to chemoembolization composition foranti-angiogenic agent delivery. The invention further relates to amethod of preparing chemoembolization composition and to the use ofchemoembolization composition in the method for treating solid tumourcancers.

BACKGROUND OF THE INVENTION

Drug eluting beads (DEBs) have been recently developed as a new mean ofdelivering chemotherapeutic agents in a targeted and controllablefashion. DEBs, used in chemoembolization therapies, such astransarterial chemo-embolization (TACE), are embolizing beads that canbe loaded with chemotherapeutic agents and that can slowly release themin the tumour vasculature with the advantage of lower systemic toxicityand sustained local activity. Thus TACE combines therapeutic effects ofperipheral arterial occlusion with the local administration ofchemotherapeutic agents. The ideal TACE scheme should allow maximum andsustained concentration of the chemotherapeutic agent within the tumourwith minimal systemic exposure combined with calibrated tumour vesselobstruction.

Recently it has been reported that DEBs have the ability to activelysequester doxorubicin hydrochloride from a solution and release it in acontrolled and sustained fashion. They have been shown to substantiallydiminish the amount of chemotherapeutic agent that reaches the systemiccirculation, thus significantly increasing the local concentration ofthe drug and the antitumoral efficacy. For example alginate beads loadedwith doxorubicin have been reported (Yao Xue Xue Bao. 2006 August; 41(8):778-83), or polyvinyl alcohol hydrogel beads modified withsulphonate groups loaded with doxorubicin have been also reported (ClinCancer Res. 2006 Apr. 15;12(8):2563-7), for chemoembolization of theliver and demonstrated a sustained delivery in vivo. Comparison of drugloading and delivery of doxorubicin and irinotecan for different beadshas been also recently reported (Jordan et al, J Vasc Int Radiol21:1084-1090, 2010). These systems use the ion-exchange properties ofthe polymer of beads to sequester cationically charged drugs such asdoxorubicin hydrochloride, and provide a method of controlled andsustained post intraarterial delivery to a specific site within thebody, in WO 2004/071495 and WO 2006/027567, beads comprisingwater-insoluble polymer, having an overall anionic charge andelectrostatically associated with the polymer an anthracycline orcamptothecin compound are disclosed. The DEBs may be used to embolizetumours, for instance a hepatocellular carcinoma. WO 2008/138758 alsodiscloses beads (microspheres) loaded with nemorubicin hydrochloride.

However, preparing drug-loaded beads is not a straightforward procedure,especially if they should be loaded with chemotherapeutic agents of lowsolubility in aqueous media. This solubility can be insufficient toreach useful therapeutic dose, such as for sorafenib tosylate. In thecase where apparent solubility is sufficient to reach useful therapeuticdose, it may appear that some delayed precipitation occurs; makingimpossible the utilization of such preparation in a clinic setup. Forexample, sunitinib base or malate can be dissolved in standard mediaused for loading anthracycline drugs, such as doxorubicine. However insuch standard solutions, sunitinib base or malate precipitate within ashort period of time which makes loading sunitinib into beads andobtaining stable solutions in drug-loaded beads very problematic. Inaddition, no liquid oral or parenteral solution of any sunitinib salt iscommercially available, that could be used to load DEBs. Only sunitinibsuspensions in acidic media have been proposed in the literature fororal route (Navid F et al, Ann Pharmacotherapy 42:962-966, 2008; SistlaA et al Drug Dev Ind Pharm 30(1):19-25, 2004), but this is not suitablefor loading DEBs. WO 2007/090897 further discloses a method for loadingbeads with water insoluble chemotherapeutic agents by using organicsolvents. However, such an approach is not adequate for extemporaneouspreparation by the medical staff.

Therefore there remains an unmet need to provide drug-loaded beads withchemotherapeutic agents of low aqueous solubility, such asanti-angiogenic agents, more particularly such as sunitinib.

SUMMARY OF THE INVENTION

The Applicants have developed a novel method for loading drug elutingbeads with anti-angiogenic agents having low aqueous solubility, such assunitinib.

Thus the present invention provides a chemoembolization composition foranti-angiogenic agent delivery characterized in that it comprises ananti-angiogenic agent loaded in anionic drug eluting beads,

-   -   wherein said anti-angiogenic agent is selected from the group        comprising sunitinib, angiostatin K1-3, arresten,        DL-α-difluoromethyl-ornithine, fumagillin, genistein,        staurosporine, (±)-thalidomide, tumstatin, axitinib, bortezomib,        bosutinib gefitinib, pazopanib, semaxanib, sorafenib,        vandetanib, vatalanib, canertinib, dovitinib, dasatinib,        erlotinib, imatinib, lapatinib, masutinib, mubitinib,        lestaurtinib, pazopanib, tandutinib, vismodegib, and    -   wherein said anionic drug eluting beads are selected from the        group comprising sulphonate-modified polyvinyl alcohol hydrogel        beads and carboxyl-modified polyvinyl alcohol-co-sodium acrylate        beads.

The present invention further provides a method of preparingchemoembolization composition for anti-angiogenic agent deliverycharacterized in that said method comprises the following steps:

-   -   a) providing drug eluting beads, selected from the group        comprising sulphonate-modified polyvinyl alcohol hydrogel beads        or carboxyl-modified polyvinyl alcohol-co-sodium acrylate beads,    -   b) preparing an aqueous solution of anti-angiogenic agent,        wherein said aqueous solution has a pH of 2.5 to 5 and wherein        said anti-angiogenic agent has low aqueous solubility of less        than 5 mg/mL and being positively charged,    -   c) adding a sugar or polyol solution to the anti-angiogenic        solution of step b)    -   d) contacting anti-angiogenic solution of step c) with drug        eluting beads of step a).

The present invention also provides the chemoembolization composition ofthe invention for use in the method for treating solid tumour cancers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage of remaining drug in 2 mL of supernatantsolution having an initial concentration of 10 mg/mL sunitinib. Completeabsorption of 20 mg sunitinib per g of bead is observed already after 90min.

FIG. 2 shows the percentage of remaining drug in different volumes of 10mg/ml sunitinib solution used for incubation of 1 g DCbead. The legendon the right indicates the initial amount of drug initially present inthe incubation medium in mg, per g of DCbead.

FIG. 3 shows sunitinib released by loaded. DCbead in 30 mL of NaCl 0.9%,using the USP 4 flow-through method. Partial release is attributed tothe NaCl-drug ionic exchange mechanism. The 1.3 hr-period necessary toreach 75% of the plateau concentration indicates a gradual release ofthe drug.

FIG. 4 shows percentage of remaining supernatant concentration forQuadrasphere loading of 25 mg sunitinib per mg of dry microsphere.

FIG. 5 shows mean ALT level (UI/L) 6 and 24 hours after intra arterialadministration of 2 ml DC Beads loaded with 6 mg of sunitinib in thecommon hepatic artery in a rabbit model.

FIG. 6 shows mean AST level (UI/L) 6 and 24 hours after intra arterialadministration of 2 ml DC Beads loaded with 6 mg of sunitinib in thecommon hepatic artery in a rabbit model.

FIG. 7 Plasmatic sunitinib levels (ng/ml) after intra arterialadministration of 2 ml DC Beads loaded with 6 mg of sunitinib in thecommon hepatic artery in 9 rabbits.

FIG. 8 Plasmatic sunitinib levels (ng/ml) after oral administration of 6mg of in 3 rabbits.

FIG. 9 shows measurements of sunitinib levels (ng/ml) in the liver 6hours (n=1) and 24 hours (n=3) after administration of 2 ml DC Beadsloaded with 6 mg of sunitinib in the common hepatic artery.

FIG. 10 shows the survival curves of rabbits in the 3 groups treatedrespectively with DC Beads loaded with sunitinib 1.5 mg (group 1, n=6),DC Beads alone (group 2, n=5), and NaCl injected directly in thearterial branch supplying the left liver lobe.

FIG. 11 shows mean ALT level (UI/L) after intra arterial administrationof 0.05 ml DC Beads loaded with 1.5 mg of sunitinib in the arterialbranch supplying the left lateral lobe of the liver in a rabbit model(group 1).

FIG. 12 shows mean AST level (UI/L) after intra arterial administrationof 0.05 ml DC Beads loaded with 1.5 mg of sunitinib in the arterialbranch supplying the left lateral lobe of the liver in a rabbit model(group 1).

FIG. 13 Mean ALT level (UI/L) after intra arterial administration of0.05 ml DC Beads in the arterial branch supplying the left lateral lobeof the liver (group 2).

FIG. 14 Mean AST level (UI/L) after intra arterial administration of0.05 ml DC Beads in the arterial branch supplying the left lateral lobeof the liver (group 2).

FIG. 15 Mean ALT level (UI/L) after intra arterial administration of0.05 ml NaCl 0.9% in the arterial branch supplying the left lateral lobeof the liver (group 3).

FIG. 16 Mean AST level (UI/L) after intra arterial administration of0.05 ml NaCl 0.9% in the arterial branch supplying the left lateral lobeof the liver (group 3).

FIG. 17 Plasmatic sunitinib levels (ng/ml) after intra arterialadministration of 0.05 ml DC Beads loaded with 1.5 mg of sunitinib inthe common hepatic artery in 6 rabbits.

FIG. 18 Western blot analysis shows that embolization with DCBeads+sunitinib inhibits the activity of the RTKs and that embolisationwith bland DC Beads increases RTK activity after 24 h.

FIG. 19 Histopathological examination of tumor 15 days afterembolization with DC Beads loaded with sunitinib. Aspect of necrotictumor under light microscopy with HE staining. The tumor appears 100%necrotic.

FIG. 20 Histopathological examination of tumor 15 days afterembolization with DC Beads loaded with sunitinib. TUNEL stainingconfirms 100% necrosis

FIG. 21 Histopathological examniation of tumor after embolization withDC Beads loaded with Sunitinib showing DC Beads located in the peripheryof tumor and in portal spaces.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. The publications andapplications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

In the case of conflict, the present specification, includingdefinitions, will control. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in art to which the subject matter hereinbelongs. As used herein, the following definitions are supplied in orderto facilitate the understanding of the present invention.

The term “comprise” is generally used in the sense of include, that isto say permitting the presence of one or more features or components.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

As used herein the terms “subject” or “patient” are well-recognized inthe art, and, are used herein to refer to a mammal and most preferably ahuman. In some embodiments, the subject is a subject in need oftreatment or a subject with solid tumour cancers. In other embodiments,the subject can be a subject who has undergone tumour resection orradiotherapy and/or chemotherapy. The term does not denote a particularage or sex. Thus, adult and newborn subjects, whether male or female,are intended to be covered.

It has been demonstrated that ischemia induced by embolization causes anincrease in VEGF activity and neoangiogenesis, particularly in theperiphery of the tumour. This neoangiogenesis probably accounts for theregrowth of residual tumour cell after treatment by transarterialchemo-embolization (LACE). Therefore, the Applicants estimated that TACEcould be more efficient when associated with anti VEGF therapy, forexample the association of TACE with different anti-angiogenic agents.

The Applicants have developed a chemoembolization therapy which combinestherapeutic effects of peripheral arterial occlusion with the localadministration of an anti-angiogenic agent. Chemoembolization is acombination of local delivery of chemotherapy and a procedure calledembolization to treat solid tumour cancers. Solid tumour cancers thatmay be treated by chemoembolization are sarcomas, carcinomas andlymphomas. Solid tumours can develop in virtually any tissue or organ,such as lungs, breast, prostate, skin, liver and colon. In a preferredembodiment of the present invention, solid tumour cancers are malignanthypervascularised tumours, such as hepatoma or hepatocellular carcinoma(primary liver cancer) and metastasis (spread) to the liver from: coloncancer, breast cancer, carcinoid tumours and other neuroendocrinetumours, islet cell tumours of the pancreas, ocular melanoma, sarcomas,other vascular primary tumours in the body. Some success has beendemonstrated with patients whose cancer has spread to other areas of thebody, such as the chest, the kidney, pelvic organs, or oral cavity.Chemoembolization is most beneficial to patients whose disease ispredominately malignant hypervascularised tumours, such as liver,whether the tumour began in the liver or spread to the liver(metastasized) from another organ.

In chemoembolization, anti-cancer drugs are injected directly into theblood vessel feeding a cancerous tumour. In addition, synthetic materialcalled an embolic agent, such as beads, is placed inside the bloodvessels that supply blood to the tumour, in effect trapping thechemotherapy in the tumour.

As used herein, “injected directly into the blood vessel feeding acancerous tumour” or “placed inside the blood vessels that supply bloodto the tumour” refers to deposition of the embolic agent, such as beads,in an artery sufficiently close to the target tumour. Such depositioncan be accomplished by a number of means including, without limitation,the use of catheters and direct injection. Both of these methods ofdelivering embolic agents, such as beads, to a specific locale in apatient's body are well-known to those skilled in the art.

Depending on the number and type of tumours, chemoembolization may beused as the sole treatment or may be combined with other treatmentoptions such as surgery (tumour resection), standard chemotherapy and/orradiotherapy. For example chemoembolization can be applied before and/orafter surgery (tumour resection), standard chemotherapy and/orradiotherapy.

The term “standard chemotherapy” generally refers to a treatment of acancer using specific chemotherapeutic/chemical agents. Achemotherapeutic agent refers to a pharmaceutical agent generally usedfor treating cancer. The chemotherapeutic agents for treating cancerinterfering with DNA synthesis include, for example, cisplatin,carboplatin, etoposide, vincristine, cyclophosphamide, doxorubicin,ifosfamide, paclitaxel, gemcitabine, docetaxel, and irinotecan andplatinum-based anti-cancer agents, including cisplatin and carboplatin.Other anti cancer drugs comprise tyrosine kinase inhibitors such asgefitinib, imatinib; famesyl transferase inhibitors includinglonafarnib; inhibitors of mammalian targets of rapamycin (mTOR) such asevereolimus; inhibitors of PKC; PI3K and AKT and monoclonal antibodiestargeted against cellular receptors to signaling molecules such asbevacizumab, cetuximab, panitumumaband trastazumab.

The term “standard radiotherapy” refers to the use of ionizing radiationas part of cancer treatment to control malignant cells. Preferably theionizing radiation is x rays or γ-rays. It is also common to combineradiotherapy with surgery, chemotherapy, hormone therapy, orcombinations thereof. Most common cancer types can be usually treatedwith radiotherapy. The precise treatment intent (curative, adjuvant,neoadjuvant or palliative) will depend on the tumour type, location, andstage, as well as the general health of the subject in need thereof.

The standard chemotherapy and radiotherapy can be also the concomitantchemo-radiotherapy. The term “concomitant chemo-radiotherapy” is usedwhen these two treatments (chemotherapy and radiotherapy) are giveneither at the same time, or almost at the same time, for instance oneafter the other, or on the same day.

The present invention provides a chemoembolization composition foranti-angiogenic agent delivery characterized in that it comprises ananti-angiogenic agent loaded in anionic drug eluting beads,

-   -   wherein said anti-angiogenic agent is selected from the group        comprising sunitinib, angiostatin K1-3, arresten,        DL-α-difluoromethyl-ornithine, fumagillin, genistein,        staurosporine, (±)-thalidomide, tumstatin, axitinib, bortezomib,        bosutinib gefitinib, pazopanib, semaxanib, sorafenib,        vandetanib, vatalanib, canertinib, dovitinib, dasatinib,        erlotinib, imatinib, lapatinib, masutinib, mubitinib,        lestaurtinib, pazopanib, tandutinib, vismodegib, and    -   wherein said anionic drug eluting beads are selected from the        group comprising sulphonate-modified polyvinyl alcohol hydrogel        beads and carboxyl-modified polyvinyl alcohol-co-sodium acrylate        beads.

Preferably, said anti-angiogenic agent is sunitinib or imatinib. Alsopreferably said anionic drug eluting beads are sulphonate-modifiedpolyvinyl alcohol hydrogel beads or carboxyl-modified polyvinylalcohol-co-sodium acrylate beads. Most preferably said anionic drugeluting beads are sulphonate-modified polyvinyl alcohol hydrogel beads

In a further embodiment, the present invention also relates to achemoembolization composition for anti-angiogenic agent deliverycharacterized in that it comprises an anti-angiogenic agent, having lowaqueous solubility of less than 5 mg/mL and being positively charged,loaded in anionic drug eluting beads, wherein said anionic drug elutingbeads are selected from the group comprising sulphonate-modifiedpolyvinyl alcohol hydrogel beads and carboxyl-modified polyvinylalcohol-co-sodium acrylate beads.

The term “low aqueous solubility” refers to an anti-angiogenic agent ofthe invention, either salt or acidified free base thereof, havingsolubility in the sugar-free medium less than the therapeutical dosethat can be incorporated in the drug eluting beads (microspheres) whensaid drug eluting beads are contacted with the anti-angiogenic agentsolution. Typically, low aqueous solubility in the present applicationrefers to a compound having a solubility in water (sugar-free) which isless than or equal to 5 mg/mL, when measured at ambient temperature(i.e. 18-27° C.).

The terms “anti-angiogenic agent” and “angiogenesis inhibitor”, eithersalt of free base thereof, are used interchangeably herein and includeany agent that is capable of preventing or inhibiting the formation ofblood vessels, in the context of the present invention, specificexamples of anti-angiogenic agents include, but are not limited to,sunitinib, angiostatin K1-3, arresten, anti-angiogenic antithrombin(aaAT), canstatin, DL-α-difluoromethyl-ornithine, endostatin,fumagillin, genistein, minocycline, staurosporine, (±)-thalidomide,tumstatin, axitinib, bortezomib, bosutinib gefitinib, pazopanib,semaxanib, sorafenib, vandetanib, vatalanib, canertinib, dovitinib,dasatinib, erlotinib, imatinib, lapatinib, masutinib, mubitinib,nilotinib, lestaurtinib, pazopanib, tandutinib, vismodegib.

Preferably anti-angiogenic agents are selected from the group comprisingsunitinib, angiostatin K1-3, arresten, DL-α-difluoromethyl-ornithine,fumagillin, genistein, staurosporine, (±)-thalidomide, tumstatin,axitinib, bortezomib, bosutinib gefitinib, pazopanib, semaxanib,sorafenib, vandetanib, vatalanib, canertinib, dovitinib, dasatinib,erlotinib, imatinib, lapatinib, masutinib, mubitinib, lestaurtinib,pazopanib, tandutinib, vismodegib.

More preferably the anti-angiogenic agent is sunitinib and imatinib.

The present invention has been found to be of utility for formulatingchemoembolization compositions containing chemotherapeutic agents havinganti-angiogenic properties and low aqueous solubility. The invention isof particular utility for, for example, sunitinib.

Sunitinib is a small-molecule that inhibits multiple receptor tyrosinekinases (RTK) such as platelet-derived growth factor receptor (PDGF-Rs)and vascular endothelial growth factor receptors (VEGFRs), which play arole in both tumour angiogenesis and tumour cell proliferation.sunitinib also inhibits other RTK such as KIT (CD117) that, whenimproperly activated by mutations, drives the majority ofgastrointestinal stromal cell tumours. In these tumours, sunitinib hasbeen recommended as a second-line therapy for patients whose tumours areresistant to imatinib, or in patients who become intolerant to the drug.In addition, sunitinib also inhibits other RTKs like RET, CSF-1R, FLT3.The simultaneous inhibition of these targets therefore leads to areduction of tumour vascularization and to an increase cancer celldeath, and ultimately tumour shrinkage.

Sunitinib was approved by the FDA for the treatment of renal cellcarcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumour(GIST) on Jan. 26, 2006. Sunitinib demonstrated anti-tumour activity inpreclinical studies in models of colorectal cancer (CRC), non small celllung cancer (NSCLC), melanoma, renal cell carcinoma (RCC) and squamouscell carcinoma (SCC). Clinical activity was demonstrated inneuroendocrine, colon and breast cancer in phase II studies. Thecurrently approved starting oral dose of sunitinib is 50 mg/day, whichis sufficient to produce plasma concentrations of at least 50 ng/ml (theminimum concentration predicted from chemical- and cellular-based assaysto inhibit VEGFR and PDGFR).

The fact that sunitinib targets many different receptors, results inmany side effects. Hypertension and asthenia appear to be the mostcommon adverse effects with sunitinib. Diarrhoea, anorexia, disgeusia,stomatitis and hand and foot syndrom (HFS) are the other clinicallyrelevant side effects. HFS may require treatment discontinuation for afew days and/or dose reduction. Fatigue may be related to thedevelopment of hypothyroidism during sunitinib therapy. Dose reductionsare required in 50% of patients treated for RCC in order to manage thesignificant toxicities of this agent. Serious (grade 3 or 4) adverseevents occur in ≦10% of patients. Laboratory abnormalities associatedwith sunitinib therapy include increased lipase and amylase levels,reduced neutrophils, lymphocytes, and platelets counts. Therefore, theadministration of sunitinib via DEBs could reduce the systemic toxicitywhile maintaining local anti tumour efficacy, provided that sunitinibdoes not precipitate in drug eluting beads.

Drug eluting beads (DEBs) are spherical or substantially spherical beads(microspheres), based on a hydrophilic, albeit water-insoluble,biocompatible polymeric material, such as modified polyvinyl alcohol(PVA) or acrylic co-polymer. These polymers bear negatively chargedgroups for retaining positively charged chemotherapeutic agents, such asanti-angiogenic agents. Typically, the beads (microspheres) have sizeswhen equilibrated in water at 37° C., in the range 1-1000 μm, morepreferably in the range 50 to 500 μm, most preferably in the range100-300 μm. The diameter is preferably determined by measurement of thebeads size prior to loading with the positively charged chemotherapeuticagent.

Specific examples of drug eluting beads according to the presentinvention include, but are not limited to, sulphonate-modified polyvinylalcohol hydrogel beads and carboxyl-modified polyvinyl alcohol-co-sodiumacrylate beads.

Sulphonate-modified polyvinyl alcohol hydrogel, used to prepare beadsknown as DC Bear (Biocompatibles), is a microspherical soft deformableembolisation material FDA approved for the treatment of hypervasculartumours and arterio-venous malformations. It is composed of a polyvinylalcohol (PVA) polymer hydrogel that has been modified by the addition ofa sulphonic acid-containing component (2-acrylamido-2-methylpropanesulphonic acid), known as PVA (with N-acryloyl-aminoacetaldehydedimethyl acetal) copolymerized with 2-acrylamido-2methylpropanesulfonate sodium salt, and formulated by inverse suspensionpolymerisation into beads of varying size from 50 to 1200 μm. Thepresence of the negative charged moiety enables the beads to interactwith oppositely-charged chemotherapeutic agents. The sulphonate-modifiedpolyvinyl alcohol hydrogel beads are usually stored in a phosphatepacking solution.

Carboxyl-modified polyvinyl alcohol-co-sodium acrylate beads, known asHepasphere™ in Europe or Quadrasphere™ in the US (Biosphere Medical) areprecisely calibrated, spherical, hydrophilic, macroporous beads. Theyare supplied as a dry product that can be rehydrated before use. Theelastic properties allow the temporary deformation of the sphere, whichfacilitates the passage through small delivery systems. They allow acomplete occlusion of hypervascularized tumours or arteriovenousmalformations. The polymer is modified with carboxyl groups,facilitating cationic drug loading.

According to the present invention, BeadBlock™ beads (Biocompatibles)can be also used. These beads are also sulphonate-modified polyvinylalcohol hydrogel beads, but contain less sulphonate groups than theabove-mentioned DC Bead™.

According to the present invention, the positively (cationically)charged anti-angiogenic agent is associated with the anionic beadspreferably so as to allow controlled release of the anti-angiogenicagent over a period. This period may be from several minutes to weeks,preferably at least up to a few days, preferably up to 72 hours. Theanti-angiogenic agent is electrostatically bonded to the polymer ofbeads. The presence of anionic groups in the beads allows control ofrelease of positively (cationically) charged anti-angiogenic agent. Itis important that the anti-angiogenic agent is not covalently attachedto the polymer matrix.

The present invention further provides a method of preparingchemoembolization composition for anti-angiogenic agent deliverycharacterized in that said method comprises the following steps:

-   -   a) providing drug eluting beads, selected from the group        comprising sulphonate-modified polyvinyl alcohol hydrogel beads        or carboxyl-modified polyvinyl alcohol-co-sodium acrylate beads,    -   b) preparing an aqueous solution of anti-angiogenic agent,        wherein said aqueous solution has a pH of 2.5 to 5 and wherein        said anti-angiogenic agent has low aqueous solubility of less        than 5 mg/mL and being positively charged,    -   c) adding a sugar or polyol solution to the anti-angiogenic        solution of step b)    -   d) contacting anti-angiogenic solution of step c) with drug        eluting beads of step a).

Optionally saline solution is removed from anionic drug eluting beads instep a).

Usually step d) is carried out by suspending drug eluting beads inanti-angiogenic solution of step c). Typically drug eluting beads areleft in contact with anti-angiogenic solution during 1 to 5 hours,preferably 1 hour 30 minutes and 3 hours 30 minutes, more preferablyduring 2 hours.

Preferably, said anti-angiogenic agent is selected from the groupcomprising sunitinib, angiostatin K1-3, arresten, anti-angiogenicantithrombin (aaAT), canstatin, DL-α-difluoromethyl-ornithine,endostatin, fumagillin, genistein, minocycline, staurosporine,(±)-thalidomide, tumstatin, axitinib, bortezomib, bosutinib gefitinib,pazopanib, semaxanib, sorafenib, vandetanib, vatalanib, canertinib,dovitinib, dasatinib, erlotinib, imatinib, lapatinib, masutinib,mubitinib, nilotinib, lestaurtinib, pazopanib, tandutinib, vismodegib.

More preferably said anti-angiogenic agents are selected from the groupcomprising sunitinib, angiostatin K1-3, arresten,DL-α-difluoromethyl-ornithine, fumagillin, genistein, staurosporine,(±)-thalidomide, tumstatin, axitinib, bortezomib, bosutinib gefitinib;pazopanib, semaxanib, sorafenib, vandetanib, vatalanib, canertinib,dovitinib, dasatinib, erlotinib, imatinib, lapatinib, masutinib,mubitinib, lestaurtinib, pazopanib, tandutinib, vismodegib.

Most preferably said anti-angiogenic agent is sunitinib or imatinib.

The Applicants have developed an advantageous method for loadinganti-angiogenic agents, having low aqueous solubility of less than 5mg/mL, into drug eluting beads. According to this method,anti-angiogenic agent may be incorporated into the drug eluting beads bycontacting swollen beads suspended in a continuous liquid vehicle, suchas water, with an aqueous sugar or polyol solution of theanti-angiogenic agent, over a period of time, typically 2 hours, wherebydrug becomes absorbed into the body of the beads.

According to the present invention, the aqueous sugar solution of theanti-angiogenic agent is typically prepared by dissolving theanti-angiogenic agent in water, if necessary by acidification with anysuitable acid, in order to obtain an aqueous solution having a pH of 2.5to 5, preferably pH 3 to 4.5, Typically the concentration of theanti-angiogenic agent is in the range of 0 to 25 mg/mL, preferably 1 to25 mg/mL or 1 to 15 mg/mL, more preferably 2 to 25 mg/mL or 2 to 15mg/mL, most preferably 5 to 15 mg/mL. High concentration such as 15 to25 mg/mL of anti-angiogenic agent are also encompassed by the presentinvention. The solution is gently shaken until the complete dissolutionof the anti-angiogenic agent. Then a sugar or a polyol is added toachieve a solution with suitable osmotic pressure.

A suitable acid can be any mineral acid, for example hydrochloric acid,sulphuric acid or nitric acid, or any organic acid, for example lacticacid, acetic acid or formic acid. Preferably the acid is hydrochloricacid. For example for sunitinib, typically HCl 0.1N solution is used, inan equimolar quantity or slightly higher quantity to sunitinib, such as1-1.05 mole of HCl per 1 mole of sunitinib.

According to the present invention, a sugar or polyol is selected fromthe group comprising glucose, sucrose, dextran, mannitol, sorbitol ortrehalose. Typically sugar or polyol solution is at 2-15%, preferably 3to 7% and more preferably at 5%. Preferably sugar solution is 5% glucosesolution.

The Applicants have found that surprisingly, in presence of the sugar,preferably at 5% glucose, sunitinib solution was shown to be stable,without any precipitation, in contrast to saline or hydrochloric acidsolutions. Advantageously, this solution is isotonic to human plasma andit is safe for the parenteral applications. Noteworthy, the sugar is notlimited to glucose. Other sugars or polyol could be used such assucrose, dextran, mannitol, sorbitol or trehalose.

The swelling vehicle may subsequently be removed or, conveniently, maybe retained with the beads as part of the product.

The swollen beads may be used in swollen form in the form of slurry,i.e. without any or much liquid outside the swollen beads. Indeed, inorder to increase the efficiency of loading, it was found preferable toremove the saline solution, typically sodium phosphate solution, fromthe bead solution to leave slurry, before contacting swollen beads withthe aqueous sugar solution of the anti-angiogenic agent. Thusoptionally, the saline solution is removed from anionic drug elutingbeads in step a).

Alternatively, the drug-loaded beads can be removed from any remainingdrug loading solution and the beads dried by any of the classicaltechniques employed to dry pharmaceutical-based products. This couldinclude, but is not limited to, air drying at room or elevatedtemperatures or under reduced pressure or vacuum; classicalfreeze-drying; atmospheric pressure-freeze drying; solution enhanceddispersion of supercritical fluids (SEDS). Alternatively the drug-loadedbeads may be dehydrated using an organic solvent to replace water in aseries of steps, followed by evaporation of the more volatile organicsolvent. A solvent should be selected which is a non-solvent for thedrug.

A typical classical freeze-drying process might proceed as follows: thesample is aliquoted into partially stoppered glass vials, which areplaced on a cooled, temperature controlled shelf within the freezedryer. The shelf temperature is reduced and the sample is frozen to auniform, defined temperature. After complete freezing, the pressure inthe dryer is lowered to a defined pressure to initiate primary drying.During the primary drying, water vapour is progressively removed fromthe frozen mass by sublimation whilst the shelf temperature iscontrolled at a constant, low temperature. Secondary drying is initiatedby increasing the shelf temperature and reducing the chamber pressurefurther so that water absorbed to the semi-dried mass can be removeduntil the residual water content decreases to the desired level. Thevials can be sealed, in situ, under a protective atmosphere if required.

Atmospheric pressure freeze-drying is accomplished by rapidlycirculating very dry air over a frozen product. In comparison with theclassical freeze-drying process, freeze-drying without a vacuum has anumber of advantages. The circulating dry gas provides improved heat andmass transfer from the frozen sample. Of particular interest is the factthat by using atmospheric spray-drying processes, instead of a cake, afine, free-flowing powder is obtained. Particles can be obtained whichhave submicron diameters; this is ten-fold smaller than can be generallyobtained by milling. The particulate nature, with its high surface arearesults in an easily rehydratable product.

Although the chemoembolization composition of the invention may be madeup from beads and canonically charged chemotherapeutic agent immediatelybefore administration, it is also possible that the chemoembolizationcomposition is supplied pre-loaded with the drug. For the latter case,dried drug-loaded beads may be hydrated immediately before use.Alternatively the chemoembolization composition which is supplied may befully compounded and preferably comprises drug eluting beads withabsorbed or adsorbed anti angiogenic agent and imbibed aqueous sugarsolution.

The level of anti-angiogenic agent in the chemoembolization compositionof the invention, which is administered, is preferably in the range 0.1to 500 mg per ml composition, preferably 10 to 100 mg per ml. Preferablythe treatment is repeated one to five times and for each dose the amountof anti-angiogenic agent administered is in the range 0.1 to 100 mg perml, preferably 10 to 100 mg per ml. The amount of the chemoembolizationcomposition administered in a normal treatment is in the range 1 to 6ml. The total amount of anti-angiogenic agent administered per dose ispreferably in the range 10 to 1000 mg, more preferably 25 to 250 mg.Based on the release data as shown in the Examples below, the Applicantsbelieve this will give therapeutically effective concentrations in thetumour and that significant levels of intracellular delivery should takeplace whereby a therapeutic effect will be achieved. The adversesystemic side effects of anti-angiogenic agent administration should beavoided.

The present invention further provides the chemoembolization compositionof the invention for use in the method for treating solid tumourcancers.

In a preferred embodiment of the present invention, solid tumour cancersare malignant hypervascularised tumours, such as hepatoma orhepatocellular carcinoma (primary liver cancer) and metastasis (spread)to the liver from: colon cancer, breast cancer, GIST liver metastasis,renal cancer, carcinoid tumours and other neuroendocrine tumours, isletcell tumours of the pancreas, ocular melanoma, sarcomas, other vascularprimary tumours in the body.

Thus preferably said malignant hypervascularised tumours are selectedfrom the group comprising hepatocellular carcinoma (HCC), livermetastasis, cholangiomas, neuroendorine tumours, GIST liver metastasisand renal cancer.

The present invention also provides a method of treatment of solidtumour cancers in a subject suffering therefrom comprising administeringa therapeutically effective amount of the chemoembolization compositionof the invention. Preferably said solid tumour cancers are malignanthypervascularised tumours. More preferably said malignanthypervascularised tumours are selected from the group comprisinghepatocellular carcinoma (HCC), liver metastasis, cholangiomas,neuroendorine tumours, GIST liver metastasis and renal cancer.

The method of this invention can be used to treat any solid tumourcancer to which blood is supplied by a dedicated, relatively reachableartery such as the renal, hepatic, pulmonary and cardiac arteries. In anaspect of this invention, the solid tumour cancer is a hepatocellularcarcinoma.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder, for example solid tumour cancer, as well as those inwhich the disorder, for example solid tumour cancer, is to be prevented.Hence, the mammal, preferably human, to be treated herein may have beendiagnosed as having the disorder, for example solid tumour cancer, ormay be predisposed or susceptible to the disorder, for example solidtumour cancer, “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals orpet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably,the mammal is human.

The term “therapeutically effective amount” refers to an amount of achemoembolization composition effective to treat a disease or disorderin a mammal. In the case of solid tumour cancer, the therapeuticallyeffective amount of the chemoembolization composition may reduce thetumour size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumour metastasis; inhibit, to someextent, tumour growth; and/or relieve to some extent one or more of thesymptoms associated with the solid tumour cancer. To the extent thechemoembolization composition of the present invention may preventgrowth and/or kill existing cancer cells, it may be cytostatic and/orcytatoxic. The phrase “therapeutically effective amount” is used hereinto mean an amount sufficient to prevent, or preferably reduce by atleast about 30 percent, preferably by at least 50 percent, preferably byat least 70 percent, preferably by at least 80 percent, preferably by atleast 90%, a clinically significant change in the growth or progressionor mitotic activity of a target cellular mass, group of cancer cells orother feature of pathology.

The daily dose of the chemoembolization composition of the presentinvention will necessarily be varied depending upon the host treated,the particular route of administration, and the severity and kind of theillness being treated. Accordingly the optimum dosage may be determinedby the practitioner Who is treating any particular patient. Further, itis noted that the clinician or treating physician will know how and whento start, interrupt, adjust, or terminate therapy in conjunction withindividual patient response. For any chemoembolization composition usedin the method of the present invention, a therapeutically effective dosecan be estimated initially from cell culture assays, animal models, ormicrodosing of human subjects.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications without departing fromthe spirit or essential characteristics thereof. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.The present disclosure is therefore to be considered as in all aspectsillustrated and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples are, however, exemplary ofmethods of practising the present invention and are not intended tolimit the scope of the invention.

EXAMPLES Example 1 Sunitinib Loading in the DEBs

A vial of DCbead™ (100-300 microns diameter, Biocompatibles UK) wasused, after removing as much saline supernatant as possible (ca. 6 mL)from the vial. Approximately 2 mL of beads was left at the bottom of thevial. 4 ml of a 10 mg/ml solution of sunitinib base was prepared asfollows: to 40 mg of sunitinib base (i.e. 1.3 10⁴ mol) was added 1.4 mLof HCl 0.1 N (i.e. an 1.05 mole ratio to sunitinib). The sunitinib wasgently shaken until complete dissolution. Then, a 5% w/w glucosesolution was added to reach a final volume of 4 mL corresponding to asunitinib concentration of 10 mg/mL.

The solution was added to the DCbead™ vial, gently shaken once, and leftat room temperature for two hours. Aliquots were drawn at different timepoints for spectrophotometric titration at 430 nm. The percentage ofremaining supernatant concentration is shown of FIG. 1. Almost all(>98%) the drug disappeared from the supernatant, indicating progressiveloading into the beads. Finally, a drug amount of 20 mg sunitinib per gof DCbead™ was attained. This is designed hereafter as bead loading (inmg of sunitinib per g of beads).

Example 2 Variations of Drug Loading Protocol

The loading protocol of example 1 was repeated, adding various volume ofsunitinib solution at 10 mg/mL to the DCbead™ vial, therefore resultingin different nominal bead loadings, from 20 mg per g of DCbead™ to 85 mgper g of DCbead™. The time evolution of the supernatant concentration isillustrated in FIG. 2. The percentage of drug incorporated in the beadafter 2 hours incubation, relative to the drug contained in thesolution, decreased with increasing volume of sunitinib solution. Theseresults show that high drug payloads could be achieved in 2-hr loadingperiod.

Example 3 Drug Release Properties

DCbead were loaded as described in Example 1. Release experiment wereperformed using the USP method 4 (flow-through), with a Sotax CE 6apparatus, at 37° C., under constant flow (CY7 pump at 5 ml/min) in NaCl0.9%, with n=6 cells in parallel. Spectrophotometric determination at430 nm was used to measure drug concentration. The results are shown inFIG. 3, demonstrating a gradual release over 6 hours. The amount of drugeluted was a function of the total salt content of the receiving bath,as expected for a ion-exchange mechanism of delivery.

Example 4 Loading Sunitinib into Quadrasphere™

A vial of 25 mg of dry Quadraspheres™ 50-100 microns (Biosphere Medical)was used. To allow complete swelling of the beads, a sufficiently largevolume (10 mL) of solution has to be used, prepared as follows: to 25 mgof sunitinib base was added 0.88 g of HCl 0.1 N, i.e. an 1.05 molarratio to sunitinib. The vial was gently shaken until completedissolution. Then a 5% w/w glucose solution was added to reach a finalvolume of 10 mL corresponding to a sunitinib concentration of 2.5 mg/mL.The 10 mL of sunitinib solution was then poured in the Quadrasphere™vial containing 25 mg of dried microspheres.

Ten microliters of supernatant was withdrawn at predefined time pointsand absorbance measured at 430 nm. After 15 min, less than 1% of thedrug could be found in the supernatant. After 2 hours, 99.6% of the drugcould be loaded in the Quadrasphere. This demonstrates completeadsorption of the sunitinib by the Quadrasphere™ microspheres.

Example 5 Evaluation of the Pharmacokinetic and Toxic of SunitinibEluting Beads in the Rabbit Material and Methods Animal Model.

Adult healhy male New Zealand White Rabbits (n=12) weighing between3.2-2.8 kgs were used.

Preparation of Sunitinib Loaded DEB.

DC Beads measuring between 100-300 μm (Biocompatibles) were used. Themaximum volume of DC beads that can be administered in the rabbit liverwas estimated to approximately 0.2 ml, DC Beads were loaded at 30 mg ofsunitinib/ml. The DC beads were loaded with sunitinib by suspension for2 hours in a sunitinib solution to achieve the required concentration ofsunitinib per ml of beads. Small aliquots of 0.2 ml containing 6 mg ofsunitinib were prepared in individual seringes for embolization of thewhole liver. The stability of the drug has been previously verified byHPLC analysis, in vitro studies showed that the loaded drug can bereleased within a few hours (T_(75%)=1.3 hours) in a saline medium. Theexact kinetics and extent of release depends on saline concentration andvolume, as expected for a ionic exchange mechanism.

Experimental Protocol.

Animals were separated in 3 groups. The first two groups (groups 1 and2, n=4 and 4 respectively) received 0.2 ml of DEB+sunitinib (6 mg)administered intra arterially in the hepatic artery. Four animals(group 1) were sacrificed 6 hours after embolization and 4 animals(group 2) were sacrificed 24 hours after embolization. The third group(group 3, n=4) received sunitinib per os at a unique dose of 6 mg, whichshould yield a therapeutic plasmatic concentration (Cmax) of 45-55 ng/mLaccording to previous pharmacological studies (FDA drug online Sunitinibp78). Two animals in group 3 were sacrificed a 6 hours and 2 at 24hours.

Dosage of Liver Enzymes After Administration.

Liver enzymes were measured from blood samples obtained by a catheterplaced in the vein of the rabbit's ear, immediately before embolizationor oral administration of the drug and 6 hours. An additional bloodsample was obtained at 24 hours for group 2 and those animals sacrificedat 24 hours in group 3.

Dosage of Sunitinib After Administration.

Plasmatic sunitinib level were measured immediately before and after theend of the embolization procedure as well as 1, 2, 3, 4, 5, 6 hour afteradministration. An additional blood sample was obtained at 24 hours forgroup 2 and those animals sacrificed at 24 hours in group 3. Whole bloodsamples were collected into EDTA-K tubes and centrifugated. Plasmasamples were protected from light and stored frozen until analysis by LCMS/MS tandem mass spectrometry [16]. Determination of sunitinibconcentration in the liver was performed using the same massspectrometry method after sacrifice of the animals in the 3 groups.

Results

This study focused on the in vivo pharmacokinetics of sunitinibadministered by means of DEB. Serial dosage of liver enzymes allowed toevaluate potential toxicity of this administration modality andplasmatic sunitinib levels after treatment allowed to evaluate thesystemic passage of the drug which is expected to be minimal.

Liver Function Evaluation:

Administration of sunitinib/DC Beads caused a significant elevation ofALT, AST compatible with cytolysis as is usual after TACE in the liver.Bilirubin plasmatic levels were not affected by the treatment stayingbelow the detection threshold in all measurements. Administration ofsunitinib p.o. did not cause any alteration of liver enzymes

Plasmatic Sunitinib Level:

After administration of DC Beads+sunitinib in the hepatic artery,plasmatic sunitinib remained <50 ng/ml (minimum concentration predictedfrom chemical- and cellular-based assays to inhibit VEGFR and PDGFR).

Sunitinib Level in the Hepatic Tissue:

Sunitinib levels in the liver tissue were performed 6 hours (n=1) and 24hours (n=3) after aminitration of 2 ml DC Beads loaded with 6 mg ofsunitinib in the common hepatic artery. Four samples were obtained fromeach liver to avoid differences due to inhomogeneous perfusion of theliver during the administration. The mean levels were 3870 ng/ml at 6hours and 4741.7 ng/ml at 24 hours which is way above the minimumconcentration predicted from chemical- and cellular-based assays toinhibit VEGFR and PDGFR.

Conclusion

The administration of sunitinib by means of loaded DC Beads directlyinjected in the hepatic artery did not elicit unexpected toxicity.Determination of sunitinib in the liver tissue after treatment showedthat the drug is effectively eluted by the DC Beads in the tissues withobtention of high concentration in liver tissue. Serial plasmaticmeasurments showed a low release (infra therapeutic) in the systemiccirculation.

Example 6 Evaluation of the Antitumoral Effect of Sunitinib ElutingBeads in a VX2 Rabbit Tumour Model

Material and method

Animal Model.

Adult male New Zealand White Rabbits (n=15) weighing between 3.0 and 3.8kgs were used.

Experimental Protocol.

The tumors were implanted in the left liver lobe of the rabbits bylaparotomy under general anesthesia according to the technique describedby Lee et al [17]. Rabbits were separated in three groups: group 1 (n=7)received intra-arterial hepatic DEB+sunitinib, group 2 (n=6) receivedintra-arterial hepatic DEB without drug and group 3 (n=6) received asham embolization with distilled water. Selective hepatic arteriographywas performed 2 weeks after tumor implantation, under generalanesthesia. The arterial anatomy, tumor staining, vascularity, size andlocation were first assessed by a common hepatic arteriography. Thensubsequent catheterization of the tumor feeding vessel was performed andthe treatment (DEB+sunitinib) was administered. One animal in each groupwas sacrificed at 24 hours and the other were kept alive untile the15^(th) day.

Preparation of Sunitinib Loaded DEB.

The same DC Beads measuring between 100-300 μm (Biocompatibles) wereused in this experiment. DC beads were loaded with sunitinib bysuspension for 2 hours in a sunitinib solution to achieve aconcentration of 30 mg/ml. Small aliquots of 0.05 ml of DC Beadscontaining 1.5 mg of sunitinib were prepared for supra selectiveembolization of the tumor.

Measurments of Plasmatic Liver Enzymes After Administration.

Liver enzymes were measured from blood samples obtained by a catheterplaced in the vein of the rabbit's ear, immediately before and afteradministration, and every day until the 7^(th) day, One additional bloodsample was obtained at the 15^(th) day in those animals still alive.

Measurment of Plasmatic Sunitinib Levels After Administration.

Blood samples were collected to determine circulating levels (i.e.“systemic exposure”) of sunitinib. Samples were collected from acatheter placed in the vein of the rabbit's ear immediately before andafter administration, and every day until the 7^(th) day. One additionalblood sample was obtained at the 15^(th) day in those animals stillalive. Whole blood samples were collected into EDTA-K tubes andcentrifugated. Plasma was protected from light and stored frozen untilanalysis by a validated LC-MS/MS method. Toxicokinetic calculations wereperformed using the non-compartmental approach.

Anatomopathological Evaluation.

After sacrifice of the animals the livers were harvested. Sunitiniblevel in tumoral tissue were measured by LC MS/MS as in thepharmacokinetic study. The rest of the tumor was fixed in formaldehydefor histopathological preparation. The percentage of necrosis and MVDwere estimated by histopathological analysis using the TUNEL method andCD-31 labelling. We evaluated VEGFR-2 phosphorylation by Western Blotanalysis of tumor homogenates as an indirect read-out of VEGF activity.

Results

This study aimed to confirm the local antitumoral efficacy of sunitinibwhen administered intra-arterially by the means of DEB in VX2 carryingrabbits.

Catheterism of the arterial branch supplying the left lateral lobe ofthe liver and administration of the scheduled treatment was successfulin all animals.

Survival:

In Group 1 no animal died during follow up. In group 2 and 3, 2 and 3animals respectively died during follow-up. In group 2, one animal diedon the 3^(rd) day from gastric perforation and one animal died on the14^(th) day from respiratory insufficiency due to massive lungmetastatisation. In group 3, one animal died on the 3^(rd) day fromgastric perforation, one presented with paraplegia on the 7^(th) dayprobably due to a spine fracture during manipulation and was thereforesacrified and one died on the 9^(th) day from respiratory insufficiencydue to massive lung metastatisation. We did not observe any lungmetastasis at necropsis in animals in group 1.

Liver Function:

In group 1, administration of sunitinib/DC Beads caused significantelevation of ALT, AST occuring around the 2^(nd) and 3rd day aftertreatment. Bilirubin plasmatic levels were not affected. PAL and LDHlevel did not seem to vary in a significant manner after treatment. Ingroup 2, administration of DC Beads without sunitinib in the arterialbranch supplying the left lateral lobe of the caused an elevation ofboth AST and. ALT.Bilirubin plasmatic levels were not affected. PAL andLDH level did not seem to vary in a significant manner after treatment.

In group 3, administration NaCl in the arterial branch supplying theleft lateral lobe of the liver in group 3 did not cause any significanteffect of treatment on plasmatic liver enzymes levels.

Plasmatic Sunitinib Levels

In group 1, serial measurments of plasmatic sunitinib levels performedin group 1 showed that a peak in concentration occured immediately afteri.a. administration followed by a slow derease until the 3rd-4th day.

Plasmatic concentration of sunitinib remained <50 ng/ml (minimumconcentration predicted from chemical- and cellular-based assays toinhibit VEGFR and PDGFR).

Evaluation of RTK Phosphorylation by Western Blot:

In group 1, there was evident lack of phosphorylation of the RTK 24hours after embolization with sunitinib loaded DEC Beads. In group 2ther wasan augmentation of the RTK phosphorylation 24 hours afterembolization with bland DC Beads. No significant difference was observed15 days after treatment.

Histopathological Evaluation of antitumoral Effect:

On hisotpathological evaluation we observed that most of the tumorsharvested at 15 days were in a large proportion necrotic. It wasimpossible to differentiate necrosis from the spontaneous evolution ofthe VX2 tumor model from necrosis induced by treatment. Howeverinterestingly we did not observe any case of distant metastasis in group1 treated by sunitinib loaded beads

Conclusion

As in the previous study, the administration of sunitinib by means ofloaded DC Beads directly injected in the branch of the hepatic arterysupplying the tumor was well tolerated. This treatment seems to offer asurvival advantage in VX2 carrying rabbits and interestingly no animaltreated with DC Beads loaded with sunitinib presented distant metastasisat necropsy. We were able to demonstrate the inhibition of the RTK bywestern blot in tumors samples after embolization with DC Beads loadedwith sunitinib as there was a rise of RTK activity in tumors treated byDC Beads without sunitinib. This finding supports the concept thatembolization with particles loaded with an antiangiogenic agent could beuseful in the treatment of various hyper vascularized tumors. Furtherstudies are currently undercousre to further evaluate the antitumoraleffect of this combination in animal and in vitro models.

Example 7 Feasibility of Imatinib Loading in DC Bead

Imatinib was solubilized at pH 4.5 using HCl diluted in a glucose 5%solution. The drug was used at a concentration of 0.5 mg per mL ofsolution. DCbead (sulfonated polyvinyl alcohol beads, 100-300 micronsdiameter, 1 mL) were suspended in 5 mL of this solution and left incontact for 2 hours. Elution in saline (NaCl 0.9%) was monitored by highpressure liquid chromatography (mobile phase:water/methanol/triethylamine 64/35/1 v/v/v, pH 4.8, UV detection at 268nm). After 4 hours, approximately 40% of the drug amount present in theinitial loading solution could be recovered in the elution medium, asindicated by the area under the HPLC peaks, showing the feasibility ofloading imatinib into the hydrogel beads.

1. A method for a treating solid tumor cancer in a patient comprisingadministering to said patient a therapeutically effective amount of achemoembolization composition comprising anti-angiogenic agent and asugar or poly-ol loaded in anionic drug eluting beads; wherein saidanionic drug eluting beads comprise a hydrophilic, water-insoluble,biocompatible polymeric material bearing negatively charged groups,wherein said antiangiogenic agent is electrostatically bonded to thepolymer, wherein said anti-angiogenic agent is selected from the groupconsisting of sunitinib, angiostatin K1-3, arresten,DL-α-difluoromethyl-ornithine, fumagillin, genistein, staurosporine,(±)-thalidomide, tumstatin, axitinib, bortezomib, bosutinib gefitinib,pazopanib, semaxanib, sorafenib, vandetanib, vatalanib, canertinib,dovitinib, dasatinib, erlotinib, imatinib, lapatinib, masutinib,mubitinib, lestaurtinib, pazopanib, tandutinib and vismodegib.
 2. Themethod of claim 1, wherein said anti-angiogenic agent is selected fromthe group consisting of sunitinib, angiostatin K1-3, arresten,DL-α-difluoromethyl-ornithine, genistein, staurosporine,(±)-thalidomide, tumstatin, axitinib, bortezomib, bosutinib gefitinib,pazopanib, semaxanib, sorafenib, vandetanib, vatalanib, canertinib,dovitinib, dasatinib, erlotinib, imatinib, lapatinib, masutinib,mubitinib, lestaurtinib, pazopanib, tandutinib and vismodegib.
 3. Themethod of claim 1, wherein said anti-angiogenic agent is sunitinib. 4.The method of claim 1, wherein said anti-angiogenic agent is imatinib.5. The method of claim 1, wherein said anti-angiogenic agent isvandetinib.
 6. The method of claim 1, wherein said sugar or polyol isselected from the group consisting of glucose, sucrose, dextran,mannitol, sorbitol and trehalose.
 7. The method of claim 1, wherein saidsugar or poly-ol is glucose.
 8. The method of claim 1, wherein saidsolid tumor cancer is a malignant hypervascularised tumor.
 9. The methodof claim 8, wherein said malignant hypervascularised tumor is selectedfrom the group consisting of hepatocellular carcinoma (HCC), livermetastasis, cholangioma, neuroendorine tumor, GIST liver metastasis andrenal cancer.
 10. A method for treating a solid tumor cancer in apatient comprising administering to said patient a therapeuticallyeffective amount of a chemoembolization composition comprisinganti-angiogenic agent and a sugar or poly-ol loaded in anionic drugeluting beads; wherein said anionic drug eluting beads comprise ahydrophilic, water-insoluble, biocompatible polymeric material, bearingnegatively charged groups; where said antiangiogenic agent iselectrostatically bonded to the polymer, wherein said anti-angiogenicagent is selected from the group consisting of sunitinib, angiostatinK1-3, arresten, DL-α-ditluoromethyl-ornithine, fumagillin, genistein,staurosporine, (±)-thalidomide, tumstatin, axitinib, bortezomib,bosutinib gefitinib, pazopanib, semaxanib, sorafenib, vandetanib,vatalanib, canertinib, dovitinib, dasatinib, erlotinib, imatinib,lapatinib, masutinib, mubitinib, lestaurtinib, pazopanib, tandutinib andvismodegib; wherein said anionic drug eluting beads comprise a sugar orpoly-ol solution; and wherein said chemoembolization composition doesnot comprise precipitated drug.
 11. The method of claim 10, wherein saidanti-angiogenic agent is selected from the group consisting ofsunitinib, angiostatin K1-3, arresten, DL-α-difluoromethyl-ornithine,genistein, staurosporine, (±)-thalidomide, tumstatin, axitinib,bortezomib, bosutinib gefitinib, pazopanib, semaxanib, sorafenib,vandetanib, vatalanib, canertinib, dovitinib, dasatinib, erlotinib,imatinib, lapatinib, masutinib, lestaurtinib, pazopanib, tandutinib andvismodegib.
 12. The method of claim 10, wherein said anti-angiogenicagent is sunitinib.
 13. The method of claim 10, wherein saidanti-angiogenic agent is imatinib.
 14. The method of claim 10, whereinsaid antiangiogenic agent is vandetinib.
 15. The Method of claim 10,wherein said sugar or polyol is selected from the group consisting ofglucose, sucrose, dextran, mannitol, sorbitol and trehalose.
 16. Themethod of claim 10, wherein said sugar or poly-ol is glucose.
 17. Themethod of claim 10, wherein said solid tumor cancer is a malignanthypervascularised tumor.
 18. The method of claim 17, wherein saidmalignant hypervascularised tumor s selected from the group consistingof hepatocellular carcinoma (HCC), liver metastasis, cholangioma,neuroendorine tumor, GIST liver metastasis and renal cancer.
 19. Amethod for treating a solid tumor cancer in a patient comprisingadministering to said patient a therapeutically effective amount of achemoembolization composition comprising an anti-angiogenic agent and asugar or poly-ol loaded in anionic drug eluting beads; wherein saidanionic drug eluting beads comprise a hydrophilic, water-insoluble,biocompatible polymeric material, bearing negatively charged groups;wherein said antiangiogenic agent is electrostatically bonded to thepolymer; wherein said anti-angiogenic agent is selected from the groupconsisting of sunitinib, angiostatin K1-3, arresten,DL-α-ditluoromethyl-ornithine, fumagillin, genistein, staurosporine,(±)-thalidomide, tumstatin, axitinib, bortezomib, bosutinib gefitinib,pazopanib, semaxanib, sorafenib, vandetanib, vatalanib, canertinib,dovitinib, dasatinib, erlotinib, imatinib, lapatinib, masutinib,mubitinib, lestaurtinib, pazopanib, tandutinib and vismodegib; whereinsaid anionic drug eluting beads comprise a sugar or poly-ol solution;and wherein said anionic drug eluting beads do not comprise precipitateddrug.
 20. The method of claim 19, wherein said anti-angiogenic agent isselected from the group consisting of sunitinib, angiostatin K1-3arresten, DL-α-difluoromethyl-ornithine, genistein, staurosporine,(±)-thalidomide, tumstatin, axitinib, bortezomib, bosutinib gefitinib,pazopanib, semaxanib, sorafenib, vandetanib, vatalanib, canertinib,dovitinib, dasatinib, erlotinib, imatinib, lapatinib, masutinib,mubitinib, lestaurtinib, pazopanib, tandutinib and vismodegib.
 21. Themethod of claim 19, wherein said anti-angiogenic agent is sunitinib. 22.The method of claim 19, wherein said anti-angiogenic agent is imatinib.23. The method of claim 19, wherein said anti-angiogenic agent isvandetinib.
 24. The Method of claim 19, wherein said sugar or polyol isselected from the group consisting of glucose, sucrose, dextran,mannitol, sorbitol and trehalose.
 25. The method of claim 19, whereinsaid sugar or poly-ol is glucose.
 26. The method of claim 19, whereinsaid solid tumor cancer is a malignant hypervascularised tumor.
 27. Themethod of claim 25, wherein said malignant hypervascularised tumor isselected from the group consisting of hepatocellular carcinoma (HCC),liver metastasis, cholangioma, nouroendorine tumor, GIST livermetastasis and renal cancer.
 28. The method according to claim 1,wherein said hydrophilic, water-insoluble, biocompatible polymericmaterial is a modified polyvinyl alcohol or acrylic co-polymer.
 29. Themethod according to claim 1, wherein said hydrophilic water-insoluble,biocompatible polymeric material is selected from sulphonate-modifiedpolyvinyl alcohol hydrogel and carboxyl-modified polyvinylalcohol-co-sodium acrylate.
 30. The method according to claim 10,wherein said hydrophilic, water-insoluble, biocompatible polymericmaterial is a modified polyvinyl alcohol or acrylic co-polymer.
 31. Themethod according to claim 10, wherein said hydrophilic, water-insolublebiocompatible polymeric material is selected from sulphonate-modifiedpolyvinyl alcohol hydrogel and carboxyl-modified polyvinylalcohol-co-sodium acrylate.
 32. The method according to claim 19,wherein said hydrophilic, water-insoluble, biocompatible polymericmaterial is a modified polyvinyl alcohol or acrylic co-polymer.
 33. Themethod according to claim 19, wherein said hydrophilic water-insoluble,biocompatible polymeric material is selected from sulphonate-modifiedpolyvinyl alcohol hydrogel and carboxyl-modified polyvinylalcohol-co-sodium acrylate.